The objective of the proposed research is to characterize the biochemical pathways associated with 5-hydroxytryptamine (5-HT) metabolism in an invertebrate (Aplysia californica) nervous system and in a mammalian (rat) brain. Stable isotopically labelled substrate will be utilized to provide tracers of those pathways. Chemical analyses of both labelled and unlabeled chemical compounds will be performed by gas chromatography-mass spectrometry-multiple ion detection (GC-MS-MID) and by scanning gas chromatography-mass spectrometry of volatile derivatives of the chemical compounds involved in 5-HT metabolism. A more sensitive assay than those presently available for tryptophan-5-hydroxylase (EC 1.9.9.1.4) activity will be developed using GC-MS-MID and applied to assaying tryptophan 5-hydroxylase activity in individual identified neuronal cell bodies of Aplysia californica. The question of whether the presence of tryptophan-5-hydroxylase activity can be used as a reliable criterion for classifying a neuron as serotonergic will be resolved from these data. An effort will be made to determine whether 5-HT inactivating biochemical pathways are present in Aplysia. A gc-ms method of assaying tryptophan-5-hydroxylase activity in rat brain tissue will also be developed. It will then be applied to clarifying the relationship between tryptophan concentration and the rate of 5-HT biosynthesis and degradation in rat brain. Isotopic labelling will be utilized to determine the importance of the 5- hydroxyindolepyruvic acid pathway which shunts 5-hydroxyindole metabolism around 5-HT formation.